Hindered N-oxide esters of rapamycin

ABSTRACT

A compound of the structure ##STR1## wherein R and R 1  are each, independently, ##STR2## or hydrogen; R 2  and R 3  are each, independently, alkyl, arylalkyl , or R 2  and R 3  may be taken together to form a cycloalkyl ring; 
     R 4  is a heterocyclic N-oxide radical, which may be optionally substituted; 
     R 5  is alkyl or arylalkyl; 
     R 6  and R 7  are taken together to form a saturated N-alkyl-heterocyclic N-oxide, which may be optionally substituted; 
     k=0-1, 
     m=0-1; 
     n=1-6; 
     with the proviso that R and R 1  are not both hydrogen, which is useful as an immunosuppressive, antiinflammatory, antifungal, antiproliferative, and antitumor agent.

This is a division of application Ser. No. 08/345,972 filed Nov. 28,1994.

BACKGROUND OF THE INVENTION

This invention relates to hindered N-oxide esters of rapamycin and amethod for using them for inducing immunosuppression, and in thetreatment of transplantation rejection, graft vs. host disease,autoimmune diseases, diseases of inflammation, adult T-cellleukemia/lymphoma, solid tumors, fungal infections, andhyperproliferative vascular disorders.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. No.3,929,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48(1977)]disclosed that rapamycin is effective in the experimentalallergic encephalomyelitis model, a model for multiple sclerosis; in theadjuvant arthritis model, a model for rheumatoid arthritis; andeffectively inhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 ( 1989); R. Y. Calne et al., Lancet 1183 (1978);and U.S. Pat. No. 5,100,899].

Rapamycin has also been shown to be useful in preventing or treatingsystemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonaryinflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetesmellitus [Fifth Int. Conf. Inflamm. Res. Assoc. 121 (Abstract), (1990)],smooth muscle cell proliferation and intimal thickening followingvascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197(1992)], adult T-cell leukemia/lymphoma [European Patent Application525,960 A1], and ocular inflammation [European Patent Application532,862 A1].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble aminoacyl prodrugs ofrapamycin (U.S. Pat. No. 4,650,803). Recently, the numbering conventionfor rapamycin has been changed; therefore according to ChemicalAbstracts nomenclature, the esters described above would be at the 31-and 42- positions.

DESCRIPTION OF THE INVENTION

This invention provides derivatives of rapamycin which are useful asimmunosuppressive, antiinflammatory, antifungal, antiproliferative, andantitumor agents having the structure ##STR3## wherein R and R¹ areeach, independently, ##STR4## or hydrogen; R² and R³ are each,independently, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbons, orR² and R³ may be taken together to form a cycloalkyl ring of 3-8 carbonatoms;

R⁴ is a heterocyclic N-oxide radical of 5-12 atoms, which may beoptionally mono-, di-, or tri- substituted with a group selected fromalkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbonatoms, trifluoromethyl, trifiuoromethoxy, hydroxyalkyl of 1-6 carbonatoms, alkoxyalkyl of 2-12 carbon atoms, --SO₃ H, --PO₃ H₂, and --CO₂ H;

R⁵ is alkyl of 1-6 carbon atoms or arylalkyl of 7-10 carbon atoms;

R⁶ and R⁷ are taken together to form a saturated N-alkyl of 1-6 carbonatoms-heterocyclic N-oxide of 5-8 ring atoms, which may be optionallymono-, di-, or tri-substituted with a group selected from alkyl of 1-6carbon atoms, aroyl of 3-11 carbon atoms, and perfluoroalkyl of 1-6carbon atoms;

k=0-1,

m=0-1;

n=1-6;

with the proviso that R and R¹ am not both hydrogen.

It is preferred that the heterocyclic N-oxide radical defined in R⁴ bean unsaturated or partially saturated heterocyclic N-oxide radical of5-12 atoms having 1 ring or 2 fused rings. Preferred heterocyclicN-oxide radicals include unsaturated heterocyclic N-oxide radicals suchas 1-methyl-pyrazolyl-2-N-oxide, imidazolyl-3-N-oxide, 1,2,3-triazolyl2- or 3-N-oxide, 1,2,4-triazolyl 2- or 4-N-oxide, 1,2,5-oxadiazolylN-oxide, 1,2,3,5-oxatriazolyl N-oxide, pyridinyl N-oxide, pyridazinylN-oxide, pyrimidinyl N-oxide, pyrazinyl N-oxide, 1,3,5-triazinylN-oxide, 1,2,4-triazinyl N-oxide, 1,2,3-triazinyl N-oxide,1,2,4-diazepinyl N-oxide, 2-isobenzazolyl N-oxide, 1,5-pyrindinylN-oxide, benzpyrazolyl N-oxide, benzisoxazolyl N-oxide, benzoxazolylN-oxide, quinolinyl N-oxide, isoquinolinyl N-oxide, cinnolinyl N-oxide,quinazolinyl N-oxide, naphthyridinyl N-oxide, pyrido[3,4-b]pyridinylN-oxide. pyrido[4,3-b]pyridinyl N-oxide, pyrido[2,3-b]pyridinyl N-oxide,1,4,2-benzoxazinyl N-oxide, 2,3,1-benzoxazinyl N-oxide, carbazolylN-oxide, purinyl N-oxide, and partially saturated heterocyclic N-oxideradicals selected from the list above. All of the preferred heterocyclicN-oxide radicals contain at least one double bond. When the heterocyclicN-oxide radical is partially saturated, one or more of the olefins inthe unsaturated ring system is saturated; the partially saturatedheterocyclic N-oxide radical still contains at least one double bond.The --(CH₂)n-- sidechain can be attached to any position of theheterocyclic N-oxide radical containing a carbon or nitrogen capable offorming a bond with the --(CH₂)n-- sidechain. More preferredheterocyclic N-oxide radicals are pyridinyl N-oxide, pyrazinyl N-oxide,triazinyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, imidazolylN-oxide, pyrazolyl N-oxide, quinolinyl N-oxide, and isoquinolinylN-oxide. Pyridinyl N-oxide is the most preferred heterocyclic N-oxideradical.

It is preferred that the saturated heterocyclic N-alkyl of 1-6 carbonatoms, N-oxide of 5-8 ring atoms as defined by R⁶ and R⁷ is a N-alkyl of1-6 carbon atoms-piperidine N-oxide, N-alkyl of 1-6 carbonatoms-morpholine N-oxide, N-alkyl of 1-6 carbon atoms-piperazineN-oxide, N-alkyl of 1-6 carbon atoms-pyrazolidine N-oxide, N-alkyl of1-6 carbon atoms-imidazolidine N-oxide, or N-alkyl of 1-6 carbonatoms-pyrrolidine N-oxide group. Methyl is the preferred alkyl group.

Aroyl is defined as the radical Ar--CO-- where Ar is an aryl radical. Itis preferred that the aryl moiety of the arylalkyl group and aroyl groupis a phenyl, naphthyl, pyridinyl, quinolinyl, isoquinolinyl, furanyl,benzofuranyl, benzodioxyl, benzoxazolyl, benzoisoxazolyl, indolyl,isoxazolyl, pyrimidinyl, pyrazinyl, benzopyranyl, or benzimidazolylgroup which may be optionally mono-, di-, or tri-substituted with agroup selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbonatoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12carbon atoms, --SO3H, --PO₃ H₂, and --CO₂ H. It is more preferred thatthe aryl moiety is a phenyl group that may be optionally substituted asdescribed above. The term alkyl of 1-6 carbon atoms includes bothstraight chain as well as branched carbon chains.

Of the compounds of this invention, preferred members are those in whichR¹ is hydrogen; those in which R¹ is hydrogen and R is ##STR5##

When R is ##STR6## and R¹ is hydrogen, preferred compounds are those inwhich R⁴ are pyridinyl N-oxide, pyrazinyl N-oxide, triazinyl N-oxide,pyrimidinyl N-oxide, pyridazinyl N-oxide, imidazolyl N-oxide, pyrazolylN-oxide, quinolinyl N-oxide, or isoquinolinyl N-oxide; those in which R⁴is pyridinyl N-oxide; those in which R⁴ is pyridinyl N-oxide, and k=0;those in which R⁴ is pyridinyl N-oxide, k=0, and R² and R³ are alkyl of1-6 carbon atoms or are taken together to form a cycloalkyl ring of 3-8carbon atoms.

Compounds which contain the ester group ##STR7## at the 42- or31,42-positions can be prepared by treating a suitably substitutedcarboxylic acid with a hindered base such as LDA, followed by alkylationwith a haloalkyl-nitrogen containing heterocycle. The nitrogencontaining heterocyclic moiety can be oxidized to the correspondingN-oxide using an oxidizing agent such as m-chloroperbenzoic acid(MCPBA). The resulting alkylated acid can then be activated as a mixedanhydride, with an acylating group such as 2,4,6-trichlorobenzoylchloride. Treatment of rapamycin with the mixed anhydride under mildlybasic condition provides the desired compounds. Mixtures of 42- and31,42-esters can be separated by chromatography. This scheme is outlinedbelow. The starting acids and haloalkylheterocycles are eithercommercially available or can be prepared by standard literatureprocedures. ##STR8##

Compounds which contain the ester group ##STR9## at the 42- or31,42-positions can be prepared analogously. Mixtures of 42- and31,42-esters can be separated by chromatography.

The 31-esters of this invention can be prepared by protecting the42-alcohol of rapamycin with a protecting group, such as with atert-butyl dimethylsilyl group, followed by esterification of the31-position by the procedures described above. The preparation ofrapamycin 42-silyl ethers is described in U.S. Pat. No. B1 5,120,842,which is hereby incorporated by reference. Removal of the protectinggroup provides the 31-esterified compounds. In the case of thetert-butyl dimethylsilyl protecting group, deprotection can beaccomplished under mildly acidic conditions, such as aceticacid/water/THF. The deprotection procedure is described in Example 15 ofU.S. Pat. No. 5,118,678, which is hereby incorporated by reference.

Having the 31-position esterified and the 42-position deprotected, the42-position can be esterified using a different acylating agent than wasreacted with the 31-alcohol, to give compounds having different estersat the 31- and 42- positions. Alternatively, the 42-esterifiedcompounds, prepared as described above, can be reacted with a differentacylating agent to provide compounds having different esters at the 31-and 42-positions.

This invention also covers analogous hindered esters of other rapamycinssuch as, but not limited to, 29-demethoxyrapamycin, [U.S. Pat. No.4,375,464, 32-demethoxyrapamycin under C. A. nomenclature]; rapamycinderivatives in which the double bonds in the 1-, 3-, and/or 5-positionshave been reduced [U.S. Pat. No. 5,023,262]; 29-desmethylrapamycin [U.S.Pat. No. 5,093,339, 32-desmethylrapamycin under C. A. nomenclature];7,29-bisdesmethylrapamycin [U.S. Pat. No. 5,093,338,7,32-desmethylrapamycin under C. A. nomenclature]; and15-hydroxyrapamycin [U.S. Pat. No. 5,102,876]. This invention alsocovers hindered esters at the 31-position of 42-oxorapamycin [U.S. Pat.No. 5,023,263]. The disclosures in the above cited U.S. patents arehereby incorporated by reference.

Immunosuppressive activity for representative compounds of thisinvention was evaluated in an in vitro standard pharmacological testprocedure to measure lymphocyte proliferation (LAF) and in an in vivostandard pharmacological test procedure that measures theimmunosuppressive activity of the compound tested as well as the abilityof the compound tested to inhibit or treat transplant rejection. Theprocedures for these standard pharmacological test procedures areprovided below.

The comitogen-induced thymocyte proliferation procedure (LAF) was usedas an in vitro measure of the immunosuppressive effects ofrepresentative compounds. Briefly, cells from the thymus of normalBALB/c mice are cultured for 72 hours with PHA and IL-1 and pulsed withtritiated thymidine during the last six hours. Cells are cultured withand without various concentrations of rapamycin, cyclosporin A, or testcompound. Cells are harvested and incorporated radioactivity isdetermined. Inhibition of lymphoproliferation is assessed as percentchange in counts per minute from non-drug treated controls. For eachcompound evaluated, rapamycin was also evaluated for the purpose ofcomparison. An IC₅₀ was obtained for each test compound as well as forrapamycin. When evaluated as a comparator for the representativecompounds of this invention, rapamycin had an IC₅₀ of 2.4 nM. Theresults obtained are provided as an IC₅₀.

Representative compounds of this invention were also evaluated in an invivo test procedure designed to determine the survival time of pinchskin graft from male BALB/c donors transplanted to male C₃ H(H-2K)recipients. The method is adapted from Billingham R. E. and Medawar P.B., J. Exp. Biol. 28:385-402, (1951). Briefly, a pinch skin graft fromthe donor was grafted on the dorsum of the recipient as a allograft, andan isograft was used as control in the same region. The recipients weretreated with either varying concentrations of test compoundsintraperitoneally or orally. Rapamycin was used as a test control.Untreated recipients serve as rejection control. The graft was monitoreddaily and observations were recorded until the graft became dry andformed a blackened scab. This was considered as the rejection day. Themean graft survival time (number of days±S.D.) of the drug treatmentgroup was compared with the control group. The following table shows theresults that were obtained. Results are expressed as the mean survivaltime in days. Untreated (control) pinch skin grafts are usually rejectedwithin 6-7 days. Compounds were tested using a dose of 4 mg/kgadministered i.p. or using a dose of 40 mg/kg administered p.o.

The results obtained in these standard pharmacological test proceduresare provided following the procedure for making the specific compoundsthat were tested.

The results of these standard pharmacological test proceduresdemonstrate immunosuppressive activity both in vitro and in vivo for thecompounds of this invention. The results obtained in the LAF testprocedure indicates suppression of T-cell proliferation, therebydemonstrating the immunosuppressive activity of the compounds of thisinvention. Further demonstration of the utility of the compounds of thisinvention as immunosuppressive agents was shown by the results obtainedin the skin graftstandard pharmacological test procedure. Additionally,the results obtained in the skin graft test procedure furtherdemonstrates the ability of the compounds of this invention to treat orinhibit transplantation rejection.

Based on the results of these standard pharmacological test procedures,the compounds are useful in the treatment or inhibition oftransplantation rejection such as kidney, heart, liver, lung, bonemarrow, pancreas (islet cells), cornea, small bowel, and skinallografts, and heart valve xenografts; in the treatment or inhibitionof autoimmune diseases such as lupus, rheumatoid arthritis, diabetesmellitus, myasthenia gravis, and multiple sclerosis; and diseases ofinflammation such as psoriasis, dermatitis, eczema, seborrhea,inflammatory bowel disease, and eye uveitis.

Because of the activity profile obtained, the compounds of thisinvention also are considered to have antitumor, antifungal activities,and antiproliferative activities. The compounds of this inventiontherefore also useful in treating solid tumors, adult T-cellleukemia/lymphoma, fungal infections, and hyperproliferative vasculardiseases such as restenosis and atherosclerosis.

When administered for the treatment or inhibition of the above diseasestates, the compounds of this invention can be administered to a mammalorally, parenterally, intranasally, intrabronchially, transdermally,topically, intravaginally, or rectally.

It is contemplated that when the compounds of this invention are used asan immunosuppressive or antiinflammatory agent, they can be administeredin conjunction with one or more other immunoregulatory agents. Suchother immunoregulatory agents include, but are not limited toazathioprine, corticosteroids, such as prednisone andmethylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506,OKT-3, and ATG. By combining the compounds of this invention with suchother drugs or agents for inducing immunosuppression or treatinginflammatory conditions, the lesser amounts of each of the agents arerequired to achieve the desired effect. The basis for such combinationtherapy was established by Stepkowski whose results showed that the useof a combination of rapamycin and cyclosporin A at subtherapeutic dosessignificantly prolonged heart allograft survival time. [TransplantationProc. 23: 507 (1991)].

The compounds of this invention can be formulated neat or with apharmaceutical carrier to a mammal in need thereof. The pharmaceuticalcarrier may be solid or liquid. When formulated orally, it has beenfound that 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentrate with1,2-propylene glycol, A. Nattermann & Cie. GmbH) provides an acceptableoral formulation.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carder is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carders include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carder can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carder for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The compounds of this invention may be administered rectally in the formof a conventional suppository. For administration by intranasal orintrabronchial inhalation or insufflation, the compounds of thisinvention may be formulated into an aqueous or partially aqueoussolution, which can then be utilized in the form of an aerosol. Thecompounds of this invention may also be administered transdermallythrough the use of a transdermal patch containing the active compoundand a carrier that is inert to the active compound, is non toxic to theskin, and allows delivery of the agent for systemic absorption into theblood stream via the skin. The carrier may take any number of forms suchas creams and ointments, pastes, gels, and occlusive devices. The creamsand ointments may be viscous liquid or semisolid emulsions of either theoil-in-water or water-in-oil type. Pastes comprised of absorptivepowders dispersed in petroleum or hydrophilic petroleum containing theactive ingredient may also be suitable. A variety of occlusive devicesmay be used to release the active ingredient into the blood stream suchas a semipermiable membrane covering a reservoir containing the activeingredient with or without a carder, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

In addition, the compounds of this invention may be employed as asolution, cream, or lotion by formulation with pharmaceuticallyacceptable vehicles containing 0.1-5 percent, preferably 2%, of activecompound which may be administered to a fungally affected area.

The dosage requirements vary with the particular compositions employed,the route of administration, the severity of the symptoms presented andthe particular subject being treated. Based on the results obtained inthe standard pharmacological test procedures, projected daily dosages ofactive compound would be 0.1 gg/kg-100 mg/kg, preferably between0.001-25 mg/kg, and more preferably between 0.01-5 mg/kg. Treatment willgenerally be initiated with small dosages less than the optimum dose ofthe compound. Thereafter the dosage is increased until the optimumeffect under the circumstances is reached; precise dosages for oral,parenteral, nasal, or intrabronchial administration will be determinedby the administering physician based on experience with the individualsubject treated. Preferably, the pharmaceutical composition is in unitdosage form, e.g. as tablets or capsules. In such form, the compositionis sub-divided in unit dose containing appropriate quantities of theactive ingredient; the unit dosage forms can be packaged compositions,for example, packeted powders, vials, ampoules, prefilled syringes orsachets containing liquids. The unit dosage form can be, for example, acapsule or tablet itself, or it can be the appropriate number of anysuch compositions in package form.

The following examples illustrate the preparation and biologicalactivities of representative compounds of this invention.

EXAMPLE 1 2,2-Dimethyl-3-(3-pyridinyl) Propionic Acid

Sodium hydride (4.38 g, 110 mmol, 60% dispersion, washed 2x with hexanesand dried under N2) was suspended in THF (140 mL). To this suspensionwas added diisopropylamine (15.4 mL, 110 mmol). Isobutyric acid (9.27mL, 100 mmol) was added slowly dropwise. The resulting thick whitesuspension was heated at a gentle reflux for 20 min and was then cooledto 0° C. n-Butyllithium (40 mL, 2.5 M in hexanes) was added dropwise.The reaction was warmed to room temperature and then to 35° C. for 30min. The reaction was cooled back to 0° C. and 3-picolyl chloride wasquickly added. (The 3-picolyl chloride was obtained by neutralization ofthe hydrochloride with NaHCO₃ and extracted 3× with hexane. The hexanesolution was dried over Na₂ SO₄ and concentrated to provide the freebase (caution: lacrymator). All of the hexane was not removed as thefree base is somewhat unstable in concentrated form). The reaction wasallowed to slowly warm to room temperature and stirred overnight. Thereaction was quenched with H₂ O, the aqueous layer separated and washed2× with ether. The aqueous layer was then acidified to pH 3 with 6N HCland again washed 2× ether. The aqueous phase was neutralized with NaHCO₃and extracted 4× ethyl acetate. The organic extracts were combined,dried over Na₂ SO₄, filtered and concentrated to provide a sticky solidwhich was triturated with ethyl acetate to provide 1.02 g of the desiredproduct as a tan solid.

EXAMPLE 2 2,2-Dimethyl-3-(3-pyridinyl) Propionic Acid N-oxide

To a solution of 2,2-dimethyl-3-(3-pyridinyl) propionic acid (2.0 g,11.17 mmol) in CHCl₃ (48 mL) was added MCPBA (4.1 g, 13.07 mmol, 50%wt.). The solution was stirred for 2.5 h and then concentrated in vacuo.The residue was purified via flash column chromatography using 2-20%MeOH in CH₂ Cl₂ as eluant to provide 2,2-dimethyl-3-(3-pyridinyl)propionic acid N-oxide (1.88 g, 86%) as a white powder.

¹ H NMR (200 MHz, DMSO) δ 1.0 (s, 6 H), 2.60 (s, 2 H), 7.10 (m, 1 H),7.30 (m, 1 H), 8.00 (s, 1 H), 8.05 (d, 1 H), 12.5 (s, 1 H).

mp=177°-180° C.

EXAMPLE 3 Rapamycin 42-ester with 2,2-dimethyl-3-(3-pyridinyl) PropionicAcid N-oxide

To a solution of 2,2-dimethyl-3-(3-pyridinyl) propionic acid N-oxide(1.02 g, 5.25 mmol) in THF (36 mL) was added N,N-diisopropylethylamine(0.67g, 5.25 mmol) followed by trichlorobenzoyl chloride (1.22g, 5.02mmol). The solution was stirred for 2 h, and the solvent removed via astream of N₂. Benzene (35 mL) was added followed by rapamycin (3.0 g,3.28 mmol) and DMAP (0.64 g, 5.25 mmol). The reaction was stirredovernight and then quenched with NaHCO₃ (sat). The aqueous solution wasextracted with EtOAc, dried over Na₂ SO₄ concentrated and purified viaflash column chromatography using 1-5% MeOH in CH₂ Cl₂ as eluantfollowed by recrystallization from EtOH/H₂ O to provide 1.35 g, 38% ofthe title compound.

mp=183° C.

IR (KBr) 1100 (m), 1160 (m), 1190 (m), 1275 (m), 1300 (m), 1325 (m),1375 (m), 1450 (s), 1630 (s), 1725 (s), 2920 (s), 3420 (s);

¹ H NMR (400 MHz, CDCl₃) δ 0.80-1.95 (comp m, 21 H), 0.91 (d, superimpon comp m, J=6.81 Hz, 3 H), 0.95 (d, superimp on comp m, J=6.37 Hz, 3H), 0.99 (d, superimp on comp m, J=6.37 Hz, 3 H), 1.05 (d, superimp oncomp m, J=6.59 Hz, 3 H), 1.09 (d, superimp on comp m, J=6.81 Hz, 3 H),1.17 (s, superimp on comp m, 3 H), 1.24 (s, superimp on comp m, 3 H),1.65 (s, superimp on comp m, 3 H), 1.76 (s, superimp on comp m, 3 H),2.11 (m, 4 H), 2.32 (m, 3 H), 2.59 (d, J=6.37 Hz, 1 H), 2.76 (m, 2 H),2.87 (m, 1 H), 3.12-3.42 (comp m, 3 H), 3.14 (s, superimp on comp m, 3H), 3.33 (s, superimp on comp m, 6 H), 3.57 (m, 1 H), 3.68 (m, 1 H),3.75 (d, J=5.71 Hz, 1 H), 3.86 (m, 1 H), 4.19 (d, J=5.93 Hz, 1 H), 4.66(m, 1 H), 4.77 (s, 1 H), 5.16 (m, 1 H), 5.29 (m, 1 H), 5.41 (d, J=10.11Hz, 1 H), 5.53 (dd, J=8.79, 15.16 Hz, 1 H), 5.97 (d, J=10.55 Hz, 1 H),6.13 (dd, J=9.89, 15.16 Hz, 1 H), 6.33 (m, 2 H), 7.18 (s, 2 H), 8.10 (s,2 H);

¹³ C (100 MHz, CDCl₃) δ 10.16, 13.22, 13.66, 15.87, 15.92, 16.24, 20.66,21.49, 24.77, 25.58, 27.23, 29.63, 31.19, 32.81, 33.17, 33.77, 35.10,35.70, 38.31, 38.94, 40.19, 40.49, 40.89, 41.51, 42.74, 43.33, 44.22,46.60, 51.27, 55.89, 56.26, 57.09, 59.29, 67.18, 75.38, 76.36, 77.16,80.97, 84.30, 84.40, 84.71, 86.34, 98.46, 125.25, 126.53, 128.13,129.47, 130.17, 133.57, 135.67, 136.04, 137.34, 140.09, 140.34, 140.71,166.74, 169.25, 175.81, 192.63, 208.22, 215.29; high resolution massspectrum (negative ion FAB) m/z 1090.7 [(M-•); calcd for C₆₁ H₉₀ N₂ O₁₅: 1091.39].

Results obtained in standard pharmacological test procedures:

LAF IC₅₀ :2.9 nM

Skin graft survival: i.p.: 11.17±0.98 days; oral: 11±0.89 days.

What is claimed is:
 1. A method of treating rheumatoid arthritis in amammal in need thereof which comprises administering an antiarthritiseffective amount of a compound of the structure ##STR10## wherein R andR¹ are each, independently, ##STR11## or hydrogen; R² and R³ are each,independently, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbons, orR² and R³ may be taken together to form a cycloalkyl ring of 3-8 carbonatoms;R⁴ is a heterocyclic N-oxide radical selected from the groupconsisting of 1-methyl-pyrazolyl-2-N-oxide, imidazolyl-3-N-oxide,1,2,3-triazolyl 2- or 3-N-oxide, 1,2,4-triazolyl 2- or 4-N-oxide,1,2,5-oxadiazolyl N-oxide, 1,2,3,5-oxatriazolyl N-oxide, pyridinylN-oxide, pyridazinyl N-oxide, pyrimidinyl N-oxide, pyrazinyl N-oxide,1,3,5-triazinyl N-oxide, 1,2,4-triazinyl N-oxide, 1,2,3-triazinylN-oxide, 1,2,4-diazepinyl N-oxide, 2-isobenzazolyl N-oxide,1,5-pyrindinyl N-oxide, benzpyrazolyl N-oxide, benzisoxazolyl N-oxide,benzoxazolyl N-oxide, quinolinyl N-oxide, isoquinolinyl N-oxide,cinnolinyl N-oxide, quinazolinyl N-oxide, naphthyridinyl N-oxide,pyrido[3,4-b]pyridinyl N-oxide, pyrido[4,3-b]pyridinyl N-oxide,pyrido[2,3-b]pyridinyl N-oxide, 1,4,2-benzoxazinyl N-Oxide,2,3,1-benzoxazinyl N-oxide, carbazolyl N-oxide, and purinyl N-oxide,which may be optionally mono-, di-, or tri- substituted with a groupselected from alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms,alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of2-7 carbon atoms, trifluoromethyl, trifluoromethoxy, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, --SO.sub. 3 H, --PO₃ H₂,and --CO₂ H; R⁵ is alkyl of 1-6 carbon atoms or arylalkyl of 7-10 carbonatoms; R⁶ and R⁷ are taken together to form a saturated N-alkyl of 1-6carbon atoms-heterocyclic N-oxide of 5-8 ring atoms, which may beoptionally mono-, di-, or tri-substituted with a group selected fromalkyl of 1-6 carbon atoms, aroyl of 3-11 carbon atoms, andperfluoroalkyl of 1-6 carbon atoms; k=0-1, m=0-1; n=1-6; with theproviso that R and R¹ are not both hydrogen.